Automated hybridization/imaging device for fluorescent multiplex dna sequencing

ABSTRACT

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

[0001] This invention was made with government support under Grant No.DE-FG02-88ER60700 awarded by the U.S. Department of Energy and GrantNos. RO1-HGO0517-02 and P30-HGO00199-03 awarded by the National Centerfor Human Genome Research of the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] This invention relates generally to the field of nucleic acidhybridization on membranes. More particularly, this invention relates toa method for automated multiplex sequencing of DNA.

[0003] Large scale nucleotide sequencing initiatives, such as a projectto sequence the human genome, have created a need for increasedefficiency and productivity. J. Watson, 248 Science 44 (1990).Automation of the various steps involved in sequencing is one area inwhich gains in efficiency and productivity are being made.

[0004] Multiplex sequencing, one scheme for reducing the number ofsequencing reactions and electrophoresis steps, involves the processingof a mixture of sequencing templates followed by sequentialhybridization with selected probes. G. Church & S. Kieffer-Higgins, 240Science 185 (1988); U.S. Pat. No. 4,942,124. In this method, manysequencing templates, each carrying a short known sequence or tag, areprocessed together. A single DNA preparation yields a mixture oftemplates. Sequencing reactions are performed on the mixture in theabsence of any label, and the mixed reaction products are fractionatedby electrophoresis, transferred to a membrane, and probed sequentiallyby hybridization with labeled oligonucleotides specific for each tag.Each hybridization step reveals the nucleotide sequence of one componentof the template mixture. Between hybridizations the labeled probe isremoved to permit the next hybridization without interference from theprevious probe. The advantages of multiplex sequencing come from theparallel processing of template preparations and sequencing reactions,and the simultaneous electrophoresis of mixtures of templates. Multiplexsequencing can reduce the time, effort, and resources needed for thesesteps by about a factor of the number of is different sequencingtemplates in the mixture.

[0005] The savings made in sequencing reactions and electrophoresis bymultiplex sequencing are offset to some extent, however, by new stepsthat are unnecessary in conventional sequencing protocols. Hybridizationof the membrane is an added step that is repeated with each specificprobe. Fortunately, however, the hybridization process is automatable.P. Richterich et al., 7 Bio/Techniques 52 (1989). A remaining problem isthe acquisition of sequence data in electronic form. Automatedsequencing machines are available that detect fluorescently labeledsequencing products as they migrate through a gel. The data acquired inthis way are then interpreted by an algorithm that yields a calledsequence. Most large-scale sequencing efforts have turned toward suchmachines as the only way of obtaining sufficient efficiency.

[0006] Conventionally, hybridization probes have been labeled withradioisotopes. Although radioactive probes can detect minute gquantities of DNA, they are hazardous and unstable, and high-resolutiondirect imaging of radioactive signals is not straight-forward.Non-radioactive methods of DNA detection have been developed in recentyears. The most sensitive methods involve enzymatic conversion ofsubstrates to colored, J. Leary et al., 80 Proc. Nat'l Acad. Sci. USA4045 (1983), or chemiluminescent products, J. Voyta et al., 34 J. Clin.Chem. 1157 (1988); A. Schaap et al., 28 Tetrahedron Lett. 1159 (1987);I. Bronstein et al. 180 Anal. Biochem. 95 (1989). In this approach, anenzyme is linked to a probe, and an enzyme substrate that yields acolored or chemiluminescent product is applied to the membrane. Afterthe enzyme acts on the substrate, the result is a pattern of color orlight corresponding to the pattern of the target DNA on the membrane.Although calorimetric detection of sequence ladders has been achieved,P. Richterich et al, 7 Bio/Techniques 52 (1989), the inability to removethe colored product from the membrane precludes its use for sequentialprobing.

[0007] As currently practiced, automated DNA sequencing makes use offluorescent labels for DNA detection. L. Smith et al., 321 Nature 674(1986); W. Ansorge et al., 15 Nucleic Acids Res. 4593 (1987); J. Proberet al., 238 Science 336 (1987). In these methods fluorescence detectionoccurs while the DNA is in the gel. Under such conditions, a singlefluorescent moiety per DNA molecule is sufficient for detection.Attempts at fluorescent detection in multiplex sequencing revealed agrossly inadequate limit of detection for DNA sequencing purposes. A.Karger et al., 206 Proc. SPIE 78 (1990). Background fluorescence frommost membranes adds large quantities of noise, T. Chu et al., 13Electrophoresis 105 (1992); U.S. Pat. No. 5,112,736, so that a much moreintense signal is required to achieve an adequate signal-to-noise ratiothan is required in a gel. Low fluorescence membranes, such as aminederivatized polypropylene (e, g., U.S. Pat. No. 5,112,736), are known,however such low flourescence membranes are restricted by a limit ofdetection about 100-fold too high for multiplex sequencing and themembranes are more fragile than nylon membranes.

[0008] Chemiluminescent hybridization signals are typically imaged byexposure to X-ray film although other methods are known, such as with aCCD (charge-coupled device) camera. U.S. Pat. No. 5,162,654. However thelight output from chemiluminescence is quite low. Although enzymaticturnover results in many chemiluminescent molecules per target DNAmolecule, at most one photon is emitted for each product moleculeproduced and in practice there is only about 1 photon emitted per 10 ⁴molecules. Due to the low level of light emitted, a sensitive, low-noisedetector, such as a cryogenically cooled CCD, is required for imaging,and a long exposure time is needed. A fully automated system based onchemiluminescence could be constructed, but it would be expensive andslow.

[0009] In the most straightforward operational mode, a CCD image isacquired as a snapshot, analogous to the operation of a photographiccamera. The major advantages of digital imaging, in particular fastvisualization, high sensitivity, quantitative imaging, and computerreadable format, have been well documented. E. Ribeiro et al., 194 Anal.Biochem. 174 (1991); P. Jackson et al., 9 Electrophoresis 330 (1988); P.Jackson, 270 Biochem. J. 705 (1990); K. Chan et al., 63 Anal. Chem. 746(1991); M. Lanan et al., 31 Biopolymers 1095 (1991); M. Lanan et al., 64Anal. Chem. 1967 (1992); A. Karger et al., 1206 Proc. SPIE 78 (1990); K.Misiura et al. 18 Nucleic Acids Res. 4345 (1990); D. Pollard-Knight etal., 185 Anal.Biochem. 84 (1990); Z. Boniszewski et al., 11Eleccrophoresis 432 (1990). When compared to other methods ofvisualization, however, such as autoradiography using isotope labels andX-ray film, the most obvious limitation of CCD imaging lies in thedimensions of the sensor arrays most commonly used in analyticalapplications. Their limited size rules out the recording ofhigh-resolution electropherograms on a single frame. The large number ofbands that can be resolved by high-resolution electrophoretic methodsfar exceeds the number of bands that can be adequately sampled on arrayshaving 512 to 768 CCD elements along their long axis, such as thosereferenced above.

[0010] One solution to obtaining a CCD image with adequate sampling overthe entire surface of sequencing electropherograms is by manuallymerging partially overlapping individual frames on a computer screenusing an image analysis tool. P. Jackson, 270 Biochem. J. 705 (1990).However, this procedure is time consuming and labor intensive, and thequality of the resulting composite image is compromised bydiscontinuities.

[0011] Another solution would be to use larger CCD arrays. CCD arraysconsisting of 2048 elements square are commercially available, althoughat prices that are often prohibitive for analytical applications.Considering that several thousand data points need to be collected whenseveral hundred bands are being separated, even a state-of-the-art,4-megapixel CCD area array will fall short of the most demandingrequirements of high-resolution separations. DNA sequencing, forexample, requires sampling capability for well above 500 bands on asingle lane, translating into much more than 2048 data points.

[0012] Continuous data acquisition using an area CCD can be achieved byoperating the CCD camera in Time Delay and Integration (TDI) mode. Linescan CCD cameras are also available, but TDI mode provides greatersensitivity than line scan. TDI operation adds the capability ofcontinuous data acquisition independent of the array length. this hasbeen shown for two high-speed, fluorescence DNA sequencing formats:capillary electrophoresis, A. Karger, et al., 18 Nucleic AcidsRes. 4955(1991), and ultrathin slab gels, A. Kostichka et al., 10 Bio/Technology78 (1992). TDI mode has also been used to monitor migrating fluorescentbands in capillary electrophoresis along the length of the column with aCCD camera. J. Sweedler et al., 63 Anal. Chem. 496 (1991). A TDI systemfor fluorescence detection on membranes is needed for automation ofmultiplex sequencing.

[0013] The task of converting relative band positions into nucleotidesequence is conceptually simple, however, the 1-3% error rate of humanreaders indicates that reading is more complex in practice. Bandamplitudes and positions vary due to enzyme behavior and otherbiochemical factors, and instrumentation and handling factors, such asuneven temperature distribution. Band positions as a function offragment size typically follow either quasi-logarithmic or constantspacing rules, depending on the instrumentation, but spatial jitter andposition anomalies can be large enough to superimpose adjacent bands.Interlane band amplitudes vary, and intralane band amplitudes changeboth locally and along the length of the lane. Across a givenelectrophoretic gel, bands change width and my be tilted or take oncomplex shapes. Automated sequence readers must be able to deal with allthis variation.

OBJECTS AND SUMMARY OF THE INVENTION

[0014] It is an object of the present invention to provide a system andmethod for automated multiplex sequencing of DNA.

[0015] It is also an object of the invention to provide an apparatus forautomatic hybridization and washing of sequencing membranes, detectionand imaging of fluorescent signals, and producing a called sequence withexisting software from sequence data on the sequencing membranes.

[0016] An additional object is to provide an apparatus capable ofhandling other membrane-based detection methods such as colony andplaque hybridizations; Southern, Northern, and Western blot procedures;multiplex genotyping of simple sequence repeats; sequencing and mappingby hybridization; and dot, slot, and allele-specific oligonucleotideblot techniques.

[0017] It is another object of the invention to provide a system andmethod for detecting DNA on sequencing membranes that is compatible withautomated multiplex sequencing.

[0018] It is a further object of the invention to provide a system andmethod for imaging fluorescent signals on sequencing membranes that iscompatible with automated multiplex sequencing and which is alsoapplicable to other membrane-based detection methods.

[0019] Yet a different object is to provide a method for enzyme-linkedfluorescent detection of membrane-bound nucleic acid

[0020] These and other objects may be accomplished by a method forsequencing a nucleic acid specimen by automated multiplex sequencingcomprising the steps of (a) preparing multiplex sequencing reactionproducts, (b) separating the sequencing products according to theirsize, (c) attaching the separated sequencing products to a membrane, (d)placing the membrane in a chamber device of an integrated automatedimaging hybridization chamber system comprising an hybridization chamberdevice, means for fluid delivery to the chamber device, imaging meansfor light delivery to the membrane and image recording of fluorescenceemanating from the membrane while in the chamber device, and controllermeans for controlling the operation of the system, (e) introducing afirst oligonucleotide probe containing an enzyme binding moiety, theprobe capable of specifically hybridizing with a tag sequence on themembrane-bound sequencing products, into the chamber device by the fluiddelivery means and thereby hybridizing the probe to the fractionatedproducts, (f) introducing an enzyme into the chamber device by the fluiddelivery means and binding enzyme to the binding moiety on the firstoligonucleotide probe, (g) introducing a fluorogenic substrate into thechamber device by the fluid delivery means and contacting the enzymewith the substrate so that the substrate is converted into a fluorescentproduct, (h) illuminating the fluorescent product in the chamber devicewith a beam of light from the imaging means to excite fluorescence ofthe fluorescent product and produce a pattern of hybridization thatreflects the nucleotide sequence of the nucleic acid specimen, (i)imaging the hybridization pattern by the imaging means and storing thepattern of hybridization as digital signals, and (j) converting thedigital signals by the controller means into a linear string ofnucleotides corresponding to the nucleotide sequence of the nucleic acidspecimen.

[0021] Another aspect of the invention is adding the additional steps of(k) removing the fluorescent product from the membrane by introducing anappropriate wash solution into the chamber compartment by the fluiddelivery means, (1) introducing a second oligonucleotide probe,containing a binding moiety to which an enzyme may be bound and which isable to hybridize specifically with a tag sequence different than thetag sequence of step (e), into the chamber device by the fluid deliverymeans and hybridizing the second probe to the fractionated products,introducing an enzyme into said chamber device by the fluid deliverymeans and binding the enzyme to the binding moiety on the secondoligonucleotide probe, and repeating steps (g) through (j)

[0022] Another aspect of the invention is providing a fluorogenicsubstrate that is converted into a fluorescent product by an enzyme,wherein enzyme turnover produces many copies of the fluorescent product,the fluorescent product produces a clear pattern of hybridization withthe support-bound nucleic acid, and the fluorescent product is easilyremoved for subsequent rounds of hybridization. Illustrative of suitablefluorogenic substrates is the benzothiazole derivative,2′-(2-benzothiazolyl)-6′-hydroxybenzothiazole phosphate (BBTP)-Alkalinephosphatase catalyzes the conversion of BBTP into the fluorescentproduct BBT. BBT does not diffuse on nylon membranes thus providing asharp fluorescent image of membrane-bound DNA when illuminated with awavelength of light that excites fluorescence. BBT is easily removedfrom the membrane by washing in detergent so that subsequenthybridizations can be performed on the same membrane.

[0023] Another aspect of the invention is an automated imaginghybridization chamber for automatic hybridization and imaging ofmultiplex sequencing membranes. The automated imaging m hybridizationchamber comprises a pair of concentric nested horizontal cylinders,i.e., a sealed inner cylinder that is rotatable about an axis and asealable stationary outer cylinder having a light transparent window andentry means through which the inner cylinder may be accessed.The outersurface of the inner cylinder, containing means for attaching thesequencing membrane thereto, is spaced apart from the inner surface ofthe outer cylinder thereby forming an enclosed chamber compartment, thelower portion of which may be employed for receiving fluids (referred toas a “fluid puddle”) used in the hybridization process. The portion ofthe chamber compartment above the fluid is an air space through whichthe membrane may be visualized and subjected to light through thetransparent window in the outer cylinder. The inner cylinder isrotatable on an axle and is coupled to a stepper motor through gears anda toothed belt for causing the inner cylinder to rotate. The undersideof the outer cylinder contains a plurality of valved ports extendingthrough the cylinder wall through which fluids may be injected andremoved from the chamber without cross contamination of the fluids. Eachport is either coupled to a fluid delivery module for storing anddelivering the fluids to the chamber compartment or to drain means forremoving fluid from the chamber compartment. One type of fluid deliverymodule includes a large capacity reservoir and a metering pump.Optionally, a batch heater is included for heating the fluids beforethey are inserted into the chamber. A second type of fluid deliverymodule includes a probe reservoir, a large capacity reservoir, and adual headed peristaltic pump for mixing probes with other solutions anddelivering the fluid mixture to the chamber. Probe reservoirs can alsobe of the non-mixing type. Optionally, a refrigeration unit may beincluded to chill probe or other solutions.

[0024] Another aspect of the invention includes an imaging apparatus forimaging the pattern of hybridization on the membrane while the membraneis within the chamber. The outer cylinder advantageously includes awindow of optically transparent material through which visible light maypass. A light source appropriately positioned outside the outer cylinderrelative to the transparent window illuminates the membrane at awavelength of light for exciting Fluorescence of the fluorescent producton the membrane. A lens can be used to widen the distribution of thebeam of light. A filter removes unwanted wavelengths of light. A CCDcamera also appropriately positioned outside the outer cylinder relativeto the transparent window operating in Time Delay and Integration moderecords an image of the pattern of hybridization produced by thefluorescence. A filter removes interfering wavelengths of light frombackground sources. To generate an image, the membrane, which is mountedon the inner chamber, is moved relative to the CCD camera. This movementis caused by rotation of the inner chamber by the stepper motor and issynchronized with CCD pixel shifts in the camera. The image is storedelectronically.

[0025] The above provides an overall summary of a preferred embodimentto a complete and integrated system and method for multiplex DNAsequencing. However, portions of the integrated system and/or methodtaken separately or combined may be utilized in other embodiments. Forexample, hybridization means other than the automated chamber may beutilized in a multiplexing operation. Also, membrane bound nucleic acidsmay be detected by enzyme-linked fluorescence without using the specifichybridization chamber or the specific imaging means disclosed, Further,the disclosed hybridization chamber can be used without the disclosedimaging means and/or fluorescence system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1A shows a portion of a sequencing membrane corresponding tonucleotides 290-373 from the sequencing primer at 65 minutes, 110minutes, and 20 hours after addition of the fluorogenic substrate MUFP.

[0027]FIG. 1B shows a portion of a sequencing membrane corresponding tonucleotides 290-373 from the sequencing primer at 40 minutes, 110minutes, and 23 hours after addition of the fluorogenic substrate 5MFP.

[0028]FIG. 1C shows a portion of a sequencing membrane corresponding tonucleotides 290-373 from the sequencing primer at 40 minutes, 110minutes, and 20 hours after addition of the fluorogenic substrate BBTP.

[0029]FIG. 2 is a schematic perspective view of the automated imaginghybridization chamber system according to the present invention.

[0030]FIG. 3 shows an enlarged cross sectional view of an illustrativecheck valve contained in the automated imaging hybridization chambershown in FIG. 2.

[0031]FIG. 4 shows an end sectional view of a portion of the automatedimaging hybridization chamber shown in FIG. 2 taken along lines 4-4 ofFIG. 2.

[0032]FIG. 5 shows a plot of unprocessed one-dimensional traces of aportion of a sequencing membrane read according to the presentinvention. The line patterns represent the four deoxynucleotides: T(solid), C (dashed), G (dotted), and A (dash-dot).

[0033]FIG. 6 shows the detection limit of a membrane-bound 75-meroligonucleotide in a single direct transfer electrophoresis sequenceband wherein the 75-mer oligonucleotide was probed with a complementary25-mer oligonucleotide labeled with a single 5′ biotin and detected withstreptavidin-alkaline phosphatase and BBTP.

[0034]FIG. 7 shows the detection limit of a membrane-bound 75-meroligonucleotide in a single direct transfer electrophoresis sequenceband wherein the 75-mer oligonucleotide was labeled directly with asingle 5′ biotin and detected with streptavidin-alkaline phosphatase andBBTP.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Before the present method of automated multiplex nucleotidesequencing is disclosed and described, it is to be understood that thisinvention is not limited to the particular process steps and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and their equivalents.

[0036] As used herein, “membrane” includes thin films composed of nylon,nitrocellulose, polypropylene, and the like, as well as their functionalequivalents now known in the art or later developed. Also, other solidsupports to which a nucleic acid may be bound, hybridized and detectedby enzyme-linked fluorescence is considered a functional membraneequivalent even if not in the form of a thin film.

[0037] As used herein, “macromolecule” means a nucleic acid or proteinor their functional equivalents. For example, nucleic acid is intendedto include naturally occurring and synthetic oligonucleotides andpolynucleotides regardless of whether they contain ribose, deoxyribose,or dideoxyribose sugars or a combination thereof, and regardless ofwhether they are single stranded, double stranded, or a combinationthereof. Protein is intended to include oligopeptides and polypeptides,whether naturally occurring or synthetic.

[0038] Enzyme-Linked Fluorescent Detection of DNA

[0039] One way of increasing fluorescent light output is to make use ofenzymatic turnover to yield many fluorescent molecules per target DNAmolecule. Fluorogenic substrates, compounds exhibiting low fluorescencewhich yield highly fluorescent products when acted upon by an enzyme,are available for several enzymes. Each molecule of enzyme can catalyzethe production of many fluorescent molecules. The following fluorogenicsubstrates for calf intestinal alkaline phosphatase were tested asagents for visualization of probed DNA sequence ladders on nylonmembranes: β-methylumbelliferyl phosphate (MUFP), 5-methyl fluoresceinphosphate (5MFP), and 2′-(2-benzothiazolyl)-6′-hydroxybenzothiazolephosphate (BBTP). MUFP is commonly used in fluorescent assays foralkaline phosphatase, whereas 5MFP is a weakly fluorescent compoundthat, when hydrolyzed, yields the methyl ether of the commonfluorophore, fluorescein. BBTP, also known as “NATTOPHOS,” R. Cano etal., 12 BiolTechniques 264 (1992), is hydrolyzed by alkaline phosphataseto the highly fluorescent 2′-(2-benzothiazolyl)-6′-hydroxybenzothiazole(BBT). R. Klem & W. Marvin, Preparation and Use of FluorescentBenzothidzole Derivatives, PCT WO 90/00618 (Jan. 25, 1990). While BBT isthe preferred substrate thus far considered, the invention is notlimited to any particular chemical structure for a fluorogenicsubstrate. Any substrate which does not diffuse on the membrane ofchoice thereby providing a sharp fluorescent image of membrane-bound DNAwhen illuminated with a wavelength of light that excites fluorescence issuitable. Preferably the substrate will also be is easily removed fromthe membrane by washing in detergent so that subsequent hybridizationscan be performed on the same membrane.

EXAMPLE 1

[0040] A sequencing reaction was prepared using the dideoxynucleotidechain termination method of F. Sanger et al., 74 Proc. Nat′l Acad. SciUSA 5464 (1977), with 1 pmol of M13mp19 template DNA and T7 DNApolymerase. Single-stranded template DNA was prepared from M13mp19 phagegrown and purified from E. coli 71-18 using polyethylene glycol/NaClprecipitation followed 1y phenol/chloroform extraction and ethanolprecipitation. J. Messing et al., 9 Nucleic Acids Res. 309 (1981). Forsequencing, 2 pmol of “universal” sequencing primer and 1 pmol oftemplate DNA were annealed in a 10 μl volume containing 4 mM MnCl_(2,)80 mM Tris HCl, pH 7.6, 30 mM sodium isocitrate, 10 mM dithiothreitol,and 0.1 μM of each of the four deoxynucleoside triphosphates. Annealingwas at 65° C. for two minutes, then at 37°C. for 15 minutes, beforeaddition of 2 units of T7 DNA polymerase (Sequenase 2.0, U.S.Biochemicals). After 1 minute at room temperature, 2.5 μl of thismixture was placed into each of four microtiter wells containing 2.5 μlof the appropriate deoxy-(dNTP) and dideoxy-(ddNTP) nucleotides. μld/ddNTP mixtures contained 300 μdNTP and 1 μM of the appropriate ddNTP.The reaction was incubated at,37° C. for 8 minutes and terminated by theaddition of 6 μl of stop solution containing 98% formamide, 0.1% xylenecyanol, and 0.1% bromphenol blue, and heating on a boiling water bathfor 3 minutes.

[0041] Reaction products were loaded in three sets of four lanes each(corresponding to the four deoxynucleotides A, C. , G, and T), and werefractionated and then transferred to a nylon membrane. A 4% acrylamideslab gel (Long Ranger Gel Solution, AT BioC. hem Inc., Malvern, PA)containing 1×TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA,pH 8.3) was poured between 80 cm×35 cm glass plates using a 0.2 mm to0.1 mm reverse wedge spacer. Gels were prerun at 50 V/cm, then 0.75 μlof sequence reaction was loaded per lane. DNA fragments werefractionated at 50 V/cm with passive thermostating provided by aluminumplates clamped co both glass surfaces. The electrophoresis system was adirect transfer apparatus, F. Pohl & S. Beck, 155 Meth. Enzymol. 250 (R.Wu ed., 1987) and hereby incorporated by reference, containing a customstepper motor and controls, that pulls the membrane at a constant rateperpendicular to the bottom edge of the glass plates, thus, transferringthe DNA fragments to the membrane. Conventional methods of transferringDNA from gels to membranes, such as electroblotting as described in U.S.Pat. No. 5,112,736, would also be adequate. After electrophoresis andtransfer, the membrane was removed from the apparatus, rehydrated in 40mM sodium phosphate buffer, pH 7.6, and irradiated with a total of 225mJ/cm² of UV light to cross link the DNA fragments to the nylonmembrane. Unbound DNA was removed by washing the membrane in PBS (40 mMsodium phosphate, pH 7.6, 150 mm NaCl), 5% SDS, and then the membraneswere stored in the same buffer

EXAMPLE 2

[0042] The membrane from Example 1 was placed in an automated imaginghybridization chamber, to be described momentarily, where it was probedwith a biotinylated oligonucleotide complementary to positions 80 to 110of the ladder, and then treated with a streptavidin-alkaline phosphataseconjugate. Complete cycle time was 4.5 hours. The rotation of the innercylinder within the hybridization chamber device was set to sweep a beadof fluid across the convex surface of the membrane, by movement of themembrane through the fluid, at approximately 20 second intervals.Hybridization volumes were 50 ml total, and wash volumes were 100 mleach. Unbound probe was removed by 8 washes with phosphate bufferedsaline (PBS) containing 5% SDS. Then a 1/5000 dilution ofstreptavidin-alkaline phosphatase (Boehringer Mannheim, 1000 U/ml) wasapplied in a total volume of 40 ml of PBS, 5% SDS. The enzyme conjugatewas allowed to bind for 45 minutes. Then, unbound conjugate was removedby 1 wash with PBS, 5% SDS; 1 wash with PBS, 1% SDS; 3 washes with PBS;and 3 washes with 0.1 M diethanolamine, pH 10.0, 1 mM MgCl₂, 0.01%sodium azide.

EXAMPLE 3

[0043] The membrane from Example 2 was cut so that each of the threesubstrates could be applied to a sequencing ladder. The enzymaticreaction was started by addition of 1 ml of a fluorogenic alkalinephosphatase substrate for every 300 cm² of membrane. The stock solutionswere: MUFP-50 μg/ml in 0.1 M diethanolamine, pH 10; 5MFP-50 μg/ml in 0.1M diethanolamine, pH 10; and BBTP-600 μg/ml in 2.4 mM diethanolamine, pH10. The substrate solution was applied as an even coat on the membrane.

[0044] In this example, the membranes were imaged outside the chamber,thus membranes were then placed on glass plates and wrapped withtransparent plastic film. Fluorescence emission was excited by a 458 nmline of an argon ion laser (Lexel model 96) for 5MFP and BBTP or a longwave UV mercury lamp for MUFP. Laser light was passed through a lens towiden the beam distribution and through a 450 nm, 40 nm bandwidthbandpass filter (Melles Griot). Images were obtained by a cryogenicallycooled CH210 CCD camera (Photometrics Ltd., Tucson, Arizona) equippedwith a 384×576 pixel TH7882 (Thomson CSF) CCD array and a 50 mm f/1.2Pentax camera lens stopped down to f/22. Images were collected eitherthrough a 450 nm, 40 nm bandwidth bandpass filter (Melles Griot) (MUFP),a 3 mm OG515 (Schott) color filter (5MFP), or a 560 nm, 10 nm bandwidthbandpass filter (Oriel) (BBTP). Full length scans (approximately 24inches) of the sequence ladders were obtained using TDI mode, which isdescribed in detail below. During scanning, the blot was movedperpendicularly to the camera on a translation stage consisting of aModel 506241S rail table, MD series drive, and a MC. 3000 controllerunit(DAEDAL Inc., Harrison City, PA). Two sets of lanes were acquired at150 dots per inch in 8 minutes of scan time. Image data were downloadedfrom the camera controller frame buffer to a 286 PC-AT microcomputer,transferred to a Macintosh Quandra for conversion to a TIFF file andlane-finding, and then to a DEC. 5000 workstation for base-calling andsequence analysis, described in detail below.

[0045] In all three cases, sequencing bands were visible to the eye uponillumination within 15 minutes of substrate application. With both MUFPand 5MFP, however, the stuencing ladders, though apparent, were visiblyblurred (FIGS. 1A and 1B) even at early time points (65 and 40 minutes,respectively). The blurring is thought to be due to diffusion or bulkflow of the fluorescent product, or both. In contrast, BBTP yielded arelatively sharp ladder, FIG. 1C. Even after prolonged incubation to 20hours, the sequencing ladder developed with BBTP could be read clearly.It is thought that the fluorescent product, BBT, interacts with thenylon membrane, perhaps through a simple hydrophobic interaction, toinhibit mobility. This property of BBTP, i.e. spatial localization,makes it clearly the most preferable of the substrates tested forsequence determination. MUFP and 5MFP appear to provide sufficientfluorescent product upon dephosphorylation by alkaline phosphatase to beuseful for sequencing applications if a method of spatially localizingthe fluorescent product could be developed. Similarly, other fluorogenicsubstrates than the three tested and described here could be usedprovided the fluorescent product could be bound to the membrane to yieldsharply defined bands. The large quantities of light emitted using BBTPas substrate are easily imaged. Further, the fluorescent product, BBT,can be removed from the membrane by washing with detergent, thus makingsubsequent probing feasible. In this instance, BBT was removed from themembrane by five washes in 5% SDS, 125 mM NaCl, 25 mM sodium phosphate,pH 7.2, for five minutes each at50° C. Other detergents and washconditions may be suitable, also.

[0046] One of the advantages of generating blots from DNA sequencinggels from run-time transfer to the membrane is the constant band-to-bandspacing seen on these blots. The position of sequencing bands on directtransfer electrophoresis blots is approximately linearly related tofragment length. Deviations may occur due to compressions, the inversewedge profile of the gel, and long term drift in blotting speed. Indirect transfer electrophoresis, the band spacing on the membrane,Δs_(menbrane), is proportional to the blotting speed, v_(blotting), andto the ratio of the band spacing on the gel, Δ_(gel) , to the migrationvelocity, v_(migration) . This ratio is equal to the time intervalbetween the elution of two adjacent bands, which is approximatelyconstant over a wide range of fragments covering most of the separatedsequencing fragments.${\Delta S}_{membrane} = {\frac{{\Delta S}_{gel}}{v_{migration}}v_{blotting}}$

[0047] Direct transfer electrophoresis gives the operator control overthe band spacing on the membrane by choosing the appropriate membranevelocity, which is an important feature in the case of subsequent CCDbased imaging. It is important to note that while the operator canincrease the inter band spacing in direct transfer electrophoresis byincreasing the blotting speed, the electrophoretic resolution is notimproved by this process. Loss of band resolution due to the limitedresolution of the imaging optics is easily prevented by using directtransfer electrophoresis.

[0048] Automated Imaging Hybridization Chamber

[0049] The automated imaging hybridization chamber is an integratedprobes to membrane-bound target nucleic acids under controlledconditions. When certain fluorescent probes are used, such as theenzyme-linked probe system using BBTP described above, an image of thenucleic acid on the membrane can be obtained via the transparent windowin the outer cylinder without removing the membrane from the chambercompartment. While the chamber system described herein is not limited toa single hybridization chemistry, it was configured specifically for theuse of fluorescent probe systems. It was designed, however, to beflexible enough to permit use with other systems. Other probes, such asradioactive probes, could be used in connection with this chamber, butwould not utilize the advantage of imaging with the membrane in thechamber. Further, although the hybridization chamber system was designedspecifically for sequencing applications, it is capable of handlingother membrane-based detection applications such as colony and plaquehybridizations; Southern, Northern and Western blot procedures;multiplex genotyping of simple sequence repeats; sequencing and mappingby hybridization; and dot, slot, and allele-specific oligonucleotideblot techniques. All of the functions of the chamber system are computercontrolled and fully definable by the user. The geometry of the chamberdevice is dictated by a need for the smalles workable fluid volume, forsmall air volume, to image the pattern of nucleic acid on the membrane,and for accurate temperature control (±2° C.)

[0050] Referring now to FIG. 2, the chamber device 10 contains twoconcentric nested horizontal cylinders, an inner sealed cylinder 12having a continuous cylindrical sidewall 14 and sealed ends 16 and 18,and an outer cylinder 20 having a cylindrical sidewall 22 containing avisible light transparent window 24 and entry means 26 for gainingaccess to the exterior surface of the sidewall 14 of the inner drum. Theend 28 of outer cylinder 20 is also sealed, whereas end 30 can alsoconstitute the entry means 26, in which case end 30 is sealable with an“O” ring positioned between end 30 and sidewall 22. Inner cylinder 12 isremovable from outer cylinder 20 through the entry means 26. Themembrane 32 containing bound nucleic acid to be hybridized is mounted onthe exterior surface of the sidewall 14 of the inner cylinder 12 withstainless steel wire bails (not shown). The inner cylinder 12 isrotatable on an axle 34 such that there is a small clearance (e.g.,0.125 inch) between the outside surface of the sidewall 14 of rotatableinner cylinder 12 and the inside surface of the sidewall 22 ofstationary outer cylinder 20. The space 36 between these drum sufaces isreferred to as the “chamber compartment.” The clearance between therespective sidewalls of the inner and outer cylinders that defines thechamber compartment 36 should be of sufficient width that the innercylinder 12 can rotate within the outer cylinder 20 without the membrane32 coming in contact with the interior surface of the outer cylindersidewall 22. Disposed on one end of the axle 34 is a driven gear wheel38. The mechanism for rotating the inner cylinder 12 includes a steppermotor 40 which may be disposed on the outside of the outer cylinder 20or another stationary object such as a table or machine chassis (notshown). Coupled to the stepper motor 40 is a driving gear wheel 42. Thedriving gear wheel 42 is coupled by a toothed belt 44 to the driven gearwheel 38 mounted on the axle 34. Thus, when the stepper motor 40 causesthe driving gear wheel 42 to rotate, the belt 44 is caused to rotate thedriven gear wheel 38, which in turn rotates the inner cylinder 12 withinthe outer cylinder 20.

[0051] The hybridization process requires various fluids to be broughtinto contact with the membrane 32 in the lower portion of chambercompartment 36 and then to be removed by drainage from the chambercompartment 36 and replaced by other fluids introduced into the chambercompartment 36. It is crucial that each fluid in the chamber compartment36 be kept pure and uncontaminated by other fluids. The various fluidsare added to or removed from the chamber compartment 36 through a numberof valves 46 contained in ports 48 drilled in the bottom of the cylinder20. Into each port 48 is threaded a fluid tight spring loaded checkvalve 46 assembly, FIG. 3. These valves 46 can be actuated by fluidpressure from pumps that deliver the solutions to the chambercompartment 36, by means of solenoids, or the like. The configuration ofthe valve assemblies 46 with minimal space 47 (FIG. 3) for holdingliquid on the chamber side of the valve when the valve is closed is verysignificant because of the importance of minimizing the volume of liquidthat would be available to contaminate a subsequent step of the process.One embodiment of the chamber device 10 contains 20 such ports 48 withassociated valve assemblies 46; however, the number of ports 48 andvalve assemblies 46 is variable. The valve assembly 46 in each port 48is connected, via a hose connection end 56 of valve assembly 46, to ahose 58 extending from a fluid delivery module 82, which will bedescribed in more detail momentarily. The hoses 58 connecting fluiddelivery modules 82 with valve assemblies 46 contained in the variousports 48 have no fluid connection with one another, but may be gatheredtogether in a hose bundle 84 for convenience. The ports 48 are placed inthe bottom of the horizontal outer cylinder 20 because it is importantfor minimizing contamination of succeeding steps in the process thatthey be located within the fluid puddle 86 (FIG. 4) that accumulates inthe lower portion of the chamber compartment 36 when fluid is pumpedinto the chamber compartment 36 through the valve assemblies 46contained in outer cylinder 20. This allows subsequent wash steps tocleanse space 47 in the same way and in the same order that the membraneis treated. As shown in FIG. 4, the volume of the fluid puddle 86 issufficient to contact the membrane 32 secured to the exterior surface ofthe sidewall 14 of the inner cylinder 12 when the inner cylinder 12 isrotated. Further, the dead volume in the chamber compartment 36 abovethe check valve assemblies 46 must be kept to a minimum so that theresidue from the previous solution is removed in a succeeding washingstep and for minimizing the expense connected with the various liquids.One or more ports 48 serve as drains and contain drain valves 88configured in any suitable manner so as to be operated manually orautomatically under computer control. When drain valves 88 are opened, adrain pump 89 pumps spent fluid out of the outer cylinder 20 throughdrain hoses or lines 90 to a drain reservoir 91. Pneumatic jacks (notshown) assist draining by tilting the chamber device 10 to a positionabout 5° from horizontal so that the spent fluid accumulates over thedrain valves 88, which are advantageously located near an end of theouter cylinder 20. After draining is completed, the pneumatic jacksreturn the chamber device 10 a horizontal position.

[0052] Mounted on the outside and covering the circumference of thecylindrical sidewall of horizontal outer cylinder 20, excluding the areaoccupied by window 24 and any entry means if these are different fromthe window 24, is an adhesive film heating element 92 for controllingthe temperature of the fluids in the chamber compartment 36 and hencethe temperature of the environment of the membrane. A thermocouple (notshown) is mounted on the inner surface of the outer cylinder 20 inchamber compartment 36 for monitoring the temperature of the chambercompartment 36 so that the temperature may be regulated as needed byproportional control of the heating element 92. The heating element 92and thermocouple are connected to a computer 94 to provide continuousmonitoring and regulation of the temperature. Further description ofcomputer control of the chamber device 10 will be provided momentarily.The outer cylinder 20 has ends 28 and 30 which make the chamber device10 virtually airtight so that evaporation or other loss of fluid fromthe chamber device 10 is minimized during hybridization cycles that maylast 12 hours. The small volumes of fluid utilized in making up thefluid puddle 86, e.g., about 50-100 ml in one embodiment of the chamberdevice 10, are particularly susceptible to even small amounts of fluidloss, especially at elevated temperatures.

[0053] The fluid delivery modules 82 can be made in any suitable form;two forms are illustrated in FIG. 2. The simplest is a conventionalmetering pump 96 that delivers the larger volumes of relativelyinexpensive wash solutions used for washing the membrane 32. Theserelatively inexpensive solutions are stored in large volume reservoirs98. The more complex type of module involves a double-headed peristalticpump 100, wherein each head is coupled to the same motor but has adifferent displacement. This configuration allows the fluid deliverymodule 82 to mix a relatively small volume of concentrated solution ofmore expensive and sometimes perishable probe with other solutions Theseconcentrated fluids are stored in probe concentrate reservoirs 102 untiluse, and the other solutions are stored in large volume reservoirs 98.In the event that fluids containing perishable components are used, asolid state, proportional control refrigeration unit(not shown) may becoupled to the system for preserving the perishable components fromdegradation. Certain fluids, for example wash solutions, are moreeffective at elevated temperatures than at room temperature. Thesefluids may be heated inside the chamber compartment 36 by the heatingelement 92. However, this method of heating the solutions causes a timedelay that's multiplied by the number of solutions heated. This timedelay can be eliminated by preheating the fluids with a proportionalcontrol batch heater 104 installed in the fluid path for heating thefluids just prior to their entry into chamber compartment 36.

[0054] The integrated imaging system also advantageously contains animaging assembly 106 for obtaining an image of the pattern ofhybridization to membrane-bound nucleic acid while the membrane 32 iswithin the chamber compartment 36. The sidewall 22 of outer cylinder 20contains a window 24 of optically transparent material through which themembrane 32 mounted on the exterior surface of the sidewall 14 of inner12 may be visualized. A suitable material for the window 24 is LEXAN,although other materials may function equally well. The window 24 maybecome fogged by condensation within the chamber compartment 36, howeverthe heating element 92 defogs the window 24. Optionally, an interiorwiper (not shown) can be used to defog the window 24. The imagingapparatus assembly 106 includes a light source 108, such as a laser or atungsten-halogen lamp with an appropriate band pass filter, tailored tothe absorption wavelength of the fluorescent marker and a CCD camera 110with filter for recording the image. The light source 108 is placed anappropriate distance from the chamber device 10 and pointed so that themonochromatic light emitted by the light source 108 passes through thewindow 24 and illuminates the membrane 32. The fluorescent marker on themembrane 32 is excited by the incident light and emits a fluorescentsignal at a characteristic wavelength for the particular fluorescentmarker. The fluorescent signal is detectable by the CCD camera 110placed in a suitable location for observing the emitted fluorescence.Both the light source 108 and the CCD camera 110 are connected to thecomputer 94 for controlling their operation and storing the data thatare collected by the CCD camera 110. Focusing and filtering optics areincluded in the imaging apparatus 106 for widening the beam distributionand filtering unwanted wavelengths from both the incident light and thefluorescent signal. As shown in FIG. 2, these focusing and filteringoptics can include a water-cooled infrared filter 112 and excitationbandpass filter 114 combined with the light source 108 for illuminatingthe membrane 32, and an emission bandpass filter 116 combined with theCCD camera 110 for removing unwanted background fluorescence and lightfrom the excitation light source 108. Computer control cables 118connect the computer 94 to each element of the system that's computercontrolled: heating element 92, stepper motor 40, metering pump 96, dualhead peristaltic pump 100, drain pump 89, light source 108, CCD camera110, and pneumatic jacks (not shown).

[0055] Imaging With CCD Camera

[0056] The full image of fluorescent signals being emitted from a 36×12inch membrane upon appropriate illumination can be acquired within about1.5 minutes with a CCD camera operating in TDI mode. Fluorescent imagesare acquired by a CL-E1-2048-S CCD camera (DALSA Inc., Waterloo,Ontario, Canada) equipped with a 2048×96 pixel IL-EI-2048 TDI array(DALSA). The camera housing is mated to a custom fabricated peltiercooling system, which cools the camera to 0° C. during scanningoperations. The analog output of the CCD camera is digitized and writtento disk using an AT-MIO-16F-5 Multifunctional I/O board, which ismounted in a 486 PC-AT computer. Intermediate to the CCD camera and theI/O board is a custom designed interface board, which providescompatibility between the camera and the I/O board. Also, the I/O boardcan generate a trigger signal that's used to shift CCD pixel rows forTDI scanning. A f/1.4 50-mm focal-length Nikon photographic lens ismounted on the camera, and fluorescing membranes are imaged at anapproximate distance of 29.5 inches from the lens for 150 dots per inch(dpi) resolution or at about 17 inches for 300 dpi resolution. Whengenerating an image, the membrane mounted to the rotating inner cylinderof the hybridization device is moved across the camera's field of view.

[0057] An area CCD array is composed of two major functional components,a parallel register consisting of a two-dimensional array oflight-sensitive CCD elements, and a single-line linear output register,positioned along one side of the parallel register and connected to anon-chip amplifier. Photons striking the CCD surface generatephotoelectrons, which are trapped in the CCD element nearest to thelocation of photon incidence. After the array has been exposed to light,readout of the photo generated charge is performed by simultaneouslyshifting charge packets of all lines in parallel toward the outputregister. Having arrived in the output register, the charge packets areshifted one by one in the perpendicular direction toward the on-chipamplifier and subsequent analog to digital conversion.

[0058] When a CCD snapshot is acquired, all photo generated chargepackets are read out in an uninterrupted series of parallel transfersfollowed by serial transfers and digitization after the shutter hasclosed. No charge transfer takes place during exposure. In TDI mode, incontrast, the parallel shifting of the charge toward the output registeris under the control of an external trigger, the TDI trigger. Theparallel transfer is delayed until the arrival of a pulse on the timedelay trigger line. The controlled transfer of charge is used while thephotoactive area is exposed to light and the accumulation of photogenerated charge takes place. A line in the resulting TDI imagerepresents light from a line in the object plane that's scanned acrossthe field of view of the camera. This is made possible by synchronizingthe image scan speed and the trigger signal, which controls the pixelmigration speed. The major advantage of using the TDI mode (also truefor line scan mode) is that the number of scan lines is not limited tothe number of lines of the parallel register as in standard full-frameoperation. The limitations on the length of the TDI scan are determinedby the maximum size of the digital storage space, which can be acomputer hard disk. A further advantage of TDI mode is its ability toaverage differences in CCD pixels. Each element of an image is theresult of the incremental charge accumulations in the TDI pixels whenthe image element coincided therewith.

[0059] A TDI scan offers increased sensitivity per unit time whencompared to a line scanner equipped with a linear array, because thelights collected simultaneously on the entire photoactive surface of theCCD. Because the sensitivity of a cooled CCD in low-light-levelmeasurements is approximately proportional to the size of thephotoactive area, the sensitivity of an area CCD operated in TDI mode isincreased over the sensitivity of a linear array consisting of the sametime elements approximately by the number of scan lines that aresimultaneously monitored. W. Washkurak et al., CCD Image Sensors 198-201(DALSA Inc., Waterloo, ON, Canada, 1988).

[0060] Automated Gel Reading Software

[0061] An automated sequence reader is used to convert the digitalsignals acquired by the CCD camera comprising the pattern offluorescence from the sequencing membrane into a called sequence ofnucleotides. The sequence reader used in connection with the automatedmultiplex sequencing system disclosed herein was a base-callingalgorithm described in commonly assigned copending U.S. patentapplication Ser. No. 07/978,915, filed Nov. 19, 1992, entitled METHODSAND APPARATUS FOR ANALYSIS OF CHROMATOGRAPHIC MIGRATION PATTERNS, andhereby incorporated by reference. Other automated sequence readers arecommercially available and could be used in the presently describedsystem, such as those by ABI, Pharmacia, Milligen, Hitachi, and LiC. or.

EXAMPLE 4

[0062] A full length image of the BBTP membrane, 40 minutes afteraddition of substrate, shown in FIG. 1C was acquired with a CCD cameraoperating in TDI mode and was processed using the automatic gel readingsoftware of serial no. 07/978,915 modified to accept a positivefluorescent image rather than a negative one as is usual with imaging onX-ray film. FIG. 5 shows a portion of the unprocessed one-dimensionaltraces, obtained by averaging horizontally across several pixels at thelane centers. The BBTP membrane was up-sampled by a factor of two toproduce a 300 dots-per-inch image expected by the gel reader. Lanes werefound manually using a Macintosh program, and the data shown wereproduced by averaging the value of 15 pixels per row around the lanecenters. The individual traces have not been processed in FIG. 5, butthe traces have been slightly shifted relative to one another to obtainbetter alignment.

[0063] The automated reader yielded reasonably accurate sequence as faras the membrane was imaged, 653 nucleotides from the primer. There were8 total errors (7 deletions and one substitution) in the 581 nucleotidesdeciphered, for an error rate of 1.4%. The first error occurred 381nucleotides from the primer. Four of the eight errors occurred withinthe last 39 nucleotides and two of the remaining errors were due tocompression and false stop artifacts.

[0064] Utility

EXAMPLE 5

[0065] The utility of the automated multiplex sequencing system wasshown by sequencing a 10.5 kb plasmid subclone of the humanneurofibromatosis I genomic locus. Template DNA was prepared from amapped set of 480-nucleotide sequencing transposon inserts. Thetransposon carries a NotI site, used for mapping and biotinylation, andtwo multiplex priming sites, one at each end of the element. Plasmid DNA(1 pmol) from each template was prepared by the alkaline lysis method,J. Sambrook et al., Molecular Cloning: A Laboratory Manual 1.25 (2d ed.,1989), which is hereby incorporated by reference. Then, the DNA wasdigested with NotI according to the instructions of the manufacturer,and the NotI site was filled in with bio-11-dCTP (Enzo Diagnostics)using the Klenow fragment of E. coli DNA polymerase I. Next the DNA wasdigested with EcoRI at sites flanking the inserted DNA and thebiotinylated insert DNA was bound to 0.5 mg of M280 streptavidindynabeads (Dynal, Glen Cove, N.Y.). The bead-bound DNA was thenconverted to single-stranded template by denaturation with 0.2 N NaOHand the template was subjected to a sequencing reaction as describedabove except that a magnet was used to concentrate the bound DNA afterthe reactions were terminated. Dynabeads have an iron component thatpermits magnetic concentration of the beads and the bound DNA-Sequencingreaction products were loaded on a 4% polyacrylamide gel and subjectedto electrophoresis as described in Example 1. Fractionated DNA fragmentswere directly transferred to a nylon membrane using the custom directtransfer apparatus described above. The membrane was removed from theapparatus, rehydrated in 40 mM sodium phosphate buffer, pH 7.6, andirradiated with a total of 225 mJ/cm² of UV light to cross link the DNAfragments to the nylon membrane. Unbound DNA was removed by washing themembrane in PBS (40 mM sodium phosphate, pH 7.6, 150 mM NaC. I), 5% SDS,and then the membrane was placed in the automated imaging hybridizationchamber. The controller was programmed for automatic operation and thesteps described below proceeded automatically in a cycle time of 4.5hours.

[0066] Hybridization of a probe specific for one of the two multiplexpriming sites was begun by injecting 50 ml of pre-hybridization buffer(PBS +5% SDS) into the chamber compartment by means of the fluiddelivery module. The rotation of the inner cylinder was set to sweep abead of fluid across the concave survace of the membrane atapproximately 20 second intervals. Pre-hybridization was at50° C. for 30minutes. Then, the 24-mer oligonucleotide probe was injected to aconcentration of 25 pmol/ml. The probe was 3′ end labeled withdigoxigenin-11-dUTP and dATP in a 1:10 ratio. Hybridization was carriedout for 120 minutes. Then, the hybridization buffer was removed byautomatic opening of a drain valve and draining through a drain hose.The drain valve was closed and then wash buffer (PBS+5% SDS) wasinjected into the chamber by the fluid delivery module. Unhybridizedprobe was removed by 8 washes, each in 100 ml of wash buffer for 5minutes at 50° C. Between washes, the wash buffer was drained by openingof a drain valve and draining through a drain hose, and then the drainvalve was closed. After A removal of the unhybridized probe wascompleted, a 1/5000 dilution of anti-digoxigenin antibody-alkalinephosphatase conjugate (Boehringer Mannheim) was injected into thechamber compartment by the fluid delivery module in a total volume of 40ml of PBS containing 5% SDS and was allowed to bind to the bydigoxigenin-derivatized probe for 45 minutes at 24° C. Then, theconjugate solution was drained and unbound conjugate was removed by 1wash with PBS, 5% SDS; 1 wash with PBS, 1 SDS; 3 washes with PBS; and 3washes with 0.1 M diethanolamine, pH 10.0, 1 mM MgCl_(2 ,) 0.01 % sodiumazide.

[0067] The alkaline phosphatase reaction was begun by draining the lastwash solution and then adding 50 ml of BBTP solution (6 mg/ml in 2.4 mMdiethanolamine, pH 9.0). After 180 minutes, fluorescence emission wasexcited by the 458 nm line of an argon I ion laser (Lexel Model 96). Thelaser beam was passed through a lens to widen beam distribution andthrough a 450 nm, 40 nm band width, bandpass filter (Melles Griot).Images were obtained with a cryogenically cooled C. H210 CCD camera(Photometrics), equipped with a 384×576 pixel TH7882 (Thomson) CCD arrayand a 50 mm f/1.2 Pentax camera lens stopped down to f/22. Images werecollected through a 560 nm, 10 nm bandwidth, bandpass filter (Oriel).Full length scans of the sequence ladders were obtained in TDI mode.During scanning, the membrane was moved perpendicularly to the camera bysynchronized movement of a stepper motor-driven translation stage towhich the membrane was mounted, as controlled by the computercontroller. Two lane sets were acquired at 150 dots per inch in 8minutes of scan time. Image data were transferred to a MacIntosh Quadrafor conversion to a TIFF file and lane-finding, and then to a DEC. 5000workstation for base-calling and sequence analysis.

[0068] After acquisition of the data from probing with the first probe,the fluorescent product generated from the first probing was easilyremoved by a brief detergent wash consisting of five washes for 5minutes each in PBS containing 5% SDS at 50° C. The steps ofhybridization, binding of the enzyme conjugate, enzyme reaction, anddata acquisition were repeated with a second probe. The membranecontained 10 sets of sequencing lanes, each of which contained atwo-fold multiplexed sequence ladder. Two probings of this membranegenerated sequence ladders from 18 of the sets of lanes, yielding 5841nucleotides, or an average of about 325 nucleotides per set. Themembrane was then subjected to a third round of hybridization using theprobe used in the first hybridization, and ladders corresponding to theinitial probing were reacquired with comparable quality.

EXAMPLE 6

[0069] The same procedure is used as in Example 5 with the exceptionthat images are obtained with a peltier cooled DALSA CL-E1-2048-S CCDcamera equipped with a DALSA 2048×96 pixel IL-E1-2048 array connected toa 486 PC-AT computer via an AT-MIO-16F-5 National Instruments board thatconverts the camera's analog signal to digital and writes the datadirectly to disk. Further, the membrane remains mounted on the innercylinder of the chamber device and the membrane is moved perpendicularlyto the camera by synchronized movement of the inner cylinder by thestepper motor, as controlled by the computer controller. Full lengthimages are acquired in 1.5 minutes of scan time.

EXAMPLE 7

[0070] The sensitivity, detection limits, and efficiency ofhybridization with the automatic multiplex sequence system was estimatedas follows. A range of quantities of unlabeled and biotinylated 75-meroligonucleotides were subjected to electrophoresis in a sequencing geland then transferred to a membrane filter as described above. UnlabeledDNA was probed with a singly biotinylated oligonucleotide, butbiotinylated DNA was not probed. The membrane was then exposed tostreptavidin-alkaline phosphatase conjugate, followed by addition ofBBTP. The membrane was illuminated and imaged at three time points, asshown in FIGS. 6 and 7. Signal levels were approximately linear withtime and quantity. Signal intensities were higher when biotinylated DNAwas detected (FIG. 6) than when unlabeled DNA was later biotinylated anddetected (FIG. 7). There was a two-to three-fold difference inintensity, presumably due to incomplete binding of the DNA to themembrane, effective loss of target DNA due to cross linking and stericfactors, incomplete hybridization of biotinylated probe to bound targetDNA, or A, combinations thereof. Limits of detection, defined as thequantity of DNA yielding a peak height equal to three times the standarddeviation of the background, are calculated from least-squares estimatesof the slopes of the lines. At 1 and 4 hours after application of thesubstrate, the limits of detection for a single biotin are 5 and 1attomoles (10⁻¹⁰ moles) for direct detection and 14 and 2 attomoles fora hybridized probe, respectively. Quantities of sequencing template inthe 0.5 pmol range typically yield a few hundred attomoles ofdideoxy-terminated chain per band. Thus, the technique described hereinhas the sensitivity required for low-level multiplexing (<10reprobings), and the use of probes with multiple biotin molecules or theuse of various amplification schemes should increase the signal furtherand allow for deep levels of multiplexing.

[0071] The background signal in these measurements consists of threecomponents: intrinsic membrane fluorescence, substrate fluorescence, andfluorescence of product generated from non-specifically bound DNA orstreptavidin-alkaline phosphatase or both. Initially, membranefluorescence dominates the background signal, as determined byfluorescent measurements on membranes with and without substrate. Atlater times, fluorescent product due to non-specific enzyme binding canbecome the major background component. This same background limitsenzyme-linked chemiluminescent measurements and is very sensitive toprobing, wash conditions, and the physical handling of the membrane, ascan be seen from its macroscopic inhomogeneity. Integration of automatedprobing and detection cycles, as well as alternate probing schemes, mayhelp to minimize this type of background. The background signal due tomembrane fluorescence could be decreased by the use of a lowfluorescence membrane, T. Chu et al., 13 Electrophoresis 105 (1992);U.S. Pat. No. 5,112,736, or by the development of fluorogenic substrateswhose products excite in the near-infrared, where intrinsic membranefluorescence may be lessened. Any such change of materials would have topreserve the postulated membrane/product interaction, as well as lowsubstrate fluorescence and low non-specific binding of the enzymeconjugate.

[0072] The fluorescent signal is greater than comparablechemiluminescent signals by a factor of more than 10^(5.) The absoluteintensity of the signal is more than sufficient for imaging. To avoidsaturation of the CCD during TDI scanning of the sequence ladders, thecamera lens was stopped down to f/22, which decreases the lightintensity by a factor of 336 compared to the largest aperture, f/1.4,and neutral density filters were frequently required. Increase of theexcitation light could increase the signal intensity further by severalorders of magnitude. Even though the absolute signal intensity issufficient for simple detection, increases in signal-to-noise ratiowould still be desirable, and there may be much room for improvement.BBT was excited with 458 nm light because the argon laser was aconvenient light source. BBT excites maximally near 430 nm, and theexcitation peak is approximately twice the value of 458 nm, thus atwo-fold improvement in sensitivity might be achieved by a change inexcitation wavelength. While the details of emission filtering were notfound to be extremely critical, a more careful optimization of theoptics might be fruitful.

We claim:
 1. A method for sequencing a nucleic acid specimen byautomated multiplex sequencing comprising the steps of: (a) preparingmultiplex sequencing reaction products including at least one tagsequence in a plurality of vessels, wherein said products in each vesseldiffer in length from each other and all terminate at a fixed knownnucleotide or nucleotides, wherein said fixed known nucleotide ornucleotides in one of said vessels differ from said fixed knownnucleotide or nucleotides in another of said vessels; (b) fractionatingsaid products from each said vessel according to their size; (c)attaching said fractionated products from each said vessel to amembrane; (d) placing said membrane containing said fractionatedproducts in a chamber device of an integrated automated imaginghybridization chamber system comprising an hybridization chamber device,means for fluid delivery to said chamber device, imaging means for lightdelivery to said membrane and image recording of fluorescence emanatingfrom said membrane while in said chamber device, and controller meansfor controlling operation of said system; (e) introducing a firstoligonucleotide probe containing a binding moiety to which an enzyme maybe bound and which is able to hybridize specifically with one of saidtag sequences into said chamber device by said fluid delivery means andbringing said probe into contact with said membrane thereby hybridizingsaid probe to said fractionated products; (f) introducing an enzyme intosaid chamber device by said fluid delivery means and bringing saidenzyme into contact with said first oligonucleotide probe therebybinding the enzyme to said binding moiety on said first oligonucleotideprobe; (g) introducing a fluorogenic substrate into said chamber deviceby said fluid delivery means and bringing said substrate into contactwith said enzyme, wherein said enzyme converts said fluorogenicsubstrate into a fluorescent product and wherein the fluorescent productinteracts with the membrane and is spatially localized thereon; (h)illuminating said fluorescent product in said chamber device with a beamof light from said imaging means capable of exciting fluorescence bysaid fluorescent product, wherein said fluorescence produces a patternof hybridization that reflects the nucleotide sequence of said nucleicacid specimen; (i) imaging said pattern of hybridization by said imagingmeans, wherein said pattern of hybridization is stored as a series ofdigital signals; and (j) converting said series of digital signals bysaid controller means into a linear string of nucleotides correspondingto the nucleotide sequence of the nucleic acid specimen.
 2. The methodof claim 1 further comprising (k) removing the fluorescent product fromthe membrane by introducing an appropriate wash solution into thechamber compartment by the fluid delivery means and removing the washsolution from the chamber compartment by means for fluid removal fromsaid chamber compartment via valve means contained in the lower portionof said outer cylinder sidewall; (l) introducing a secondoligonucleotide probe, containing a binding moiety to which an enzymemay be bound and which is able to hybridize specifically with one ofsaid tag sequences different than the tag sequence of step (e), intosaid chamber device by said fluid delivery means and bringing saidsecond oligonucleotide probe into contact with said membrane, therebyhybridizing said second probe to said fractionated products; (m)introducing an enzyme into said chamber device by said fluid deliverymeans and bringing said enzyme into contact with said secondoligonucleotide probe, thereby binding the enzyme to said binding moietyon said second oligonucleotide probe; and (n) repeating steps (g)through (j).
 3. The method of claim 1 wherein step (b) further comprisesfractionating said products by gel electrophoresis.
 4. The method ofclaim 3 wherein step (c) further comprises transferring saidfractionated products to said membrane by electrophoresis.
 5. The methodof claim 4 wherein said electrophoresis is direct transferelectrophoresis.
 6. The method of claim 1 wherein the hybridizationchamber device used in step (d) comprises generally horizontal,concentrically nested inner and outer cylinders having a common axis,the inner cylinder consisting of a cylindrical sidewall having anexterior surface and sealed ends, said inner cylinder being rotatableabout said axis, the outer cylinder consisting of a stationarycylindrical sidewall having interior and exterior surfaces, atransparent window contained therein and having sealable ends, saidinner cylinder being rotatable within said outer cylinder about saidaxis, said membrane being mounted on the exterior sidewall surface ofsaid inner cylinder; said inner and outer cylinders having diameterssuch that there is a space between the respective sidewalls of saidinner and outer cylinders defining a chamber compartment of sufficientwidth that said inner cylinder can rotate within said outer cylinderwithout said membrane coming in contact with the interior surface ofsaid outer cylinder sidewall, said chamber compartment being in fluidcommunication with said means for fluid delivery via valve meanscontained in a lower portion of said outer cylinder sidewall.
 7. Themethod of claim 6 wherein said hybridization chamber device furthercomprises means for rotating said inner cylinder.
 8. The method of claim7 wherein said rotating means comprises a stepper motor.
 9. The methodof claim 6 wherein said fluid delivery means comprises a plurality ofreservoirs for storing fluids and metering pumps for delivering fluid inselectable volumes.
 10. The method of claim 9 wherein said fluiddelivery means further comprises a batch heater for heating said fluidsto a selected temperature before said fluids are delivered to saidchamber compartment.
 11. The method of claim 9 wherein said fluiddelivery means further comprises a probe concentrate reservoir forstoring a concentrated liquid hybridization probe and the like, andmeans for mixing said concentrated hybridization probe and a fluid toform a hybridization solution and delivering said hybridization solutionto said chamber compartment.
 12. The method of claim 11 wherein saidfluid delivery means further comprises means for chilling saidhybridization probe.
 13. The method of claim 6 wherein saidhybridization chamber device further comprises aheating element forheating a fluid contained in said chamber compartment to a selectedtemperature.
 14. The method of claim 13 wherein said hybridizationchamber device further comprises a thermocouple for monitoring thetemperature of a fluid contained in said chamber compartment.
 15. Themethod of claim 6 further comprising means for fluid removal from saidchamber compartment via valve means contained in the lower portion ofsaid outer cylinder sidewall.
 16. The method of claim 6 wherein saidimaging means includes a light source for producing and delivering abeam of light of an appropriate wavelength to the membrane for excitingfluorescence of said fluorescent product.
 17. The method of claim 16wherein said light source comprises a laser.
 18. The method of claim 16wherein said light source comprises a tungsten-halogen lamp with a bandpass filter.
 19. The method of claim 16 wherein said imaging meansfurther comprises a filter for removing undesirable wavelengths of lightfrom said beam of light.
 20. The method of claim 16 wherein said imagingmeans includes a CCD camera for recording an image of said fluorescenceemanating from said fluorescent product on said membrane and a filterfor removing interfering wavelengths of light from said fluorescence ofsaid fluorescent product.
 21. The method of claim 20 wherein said CCDcamera is operable in Time Delay and Integration mode.
 22. The method ofclaim 1 wherein step (f) further comprises attaching to said enzyme amolecule having an affinity for binding to said binding moiety, saidmolecule forming a bridge for binding of said enzyme to said bindingmoiety.
 23. The method of claim 22 wherein said binding moiety comprisesa biotin molecule covalently attached to said probe and said bridgingmolecule is selected from the group consisting of streptavidin andavidin.
 24. The method of claim 22 wherein said binding moiety comprisesa digoxigenin molecule covalently attached to said probe and saidbridging molecule comprises an anti-digoxigenin antibody.
 25. The methodof claim 1 wherein the enzyme of step (f) is alkaline phosphatase. 26.The method of claim 1 wherein the fluorogenic substrate of step (g) is aphosphorylated benzothiazole derivative.
 27. The method of claim 26wherein the fluorogenic substrate is BBTP.
 28. The method of claim 1wherein step (i) further comprises scanning the full length of themembrane.
 29. The method of claim 28 wherein said membrane is moved insynchrony with the scanning of the membrane, said movement actuated bythe stepper motor coupled to the inner cylinder of the hybridizationchamber device and controlled by the controller means.
 30. The method ofclaim 1 wherein step (j) comprises converting said digital signals intoa linear string of nucleotides corresponding to the nucleotide sequenceof the nucleic acid specimen by an automated sequence reader.
 31. Anintegrated automated imaging hybridization chamber system for sequencinga nucleic acid specimen by automated multiplex sequencing comprising anhybridization chamber device for mounting a membrane containingfractionated multiplex sequencing reaction products, means for fluiddelivery to said chamber device, imaging means for light delivery tosaid membrane and image recording of fluorescence emanating from saidmembrane while in said chamber device, and controller means forcontrolling operation of the system.
 32. The system of claim 31 whereinthe hybridization chamber device comprises generally horizontal,concentrically nested inner and outer cylinders having a common axis,the inner cylinder consisting of a cylindrical sidewall having anexterior surface and sealed ends, said inner cylinder being rotatableabout said axis, the outer cylinder consisting of a stationarycylindrical sidewall having interior and exterior surfaces, atransparent window contained therein and having sealable ends, saidinner cylinder being rotatable within said outer cylinder about saidaxis, said membrane being mounted on the exterior sidewall surface ofsaid inner cylinder; said inner and outer cylinders having diameterssuch that there is a space between the respective sidewalls of saidinner and outer cylinders defining a chamber compartment of sufficientwidth that said inner cylinder can rotate within said outer cylinderwithout said membrane coming in contact with the interior surface ofsaid outer cylinder sidewall, said chamber compartment being in fluidcommunication with said means for fluid delivery via valve meanscontained in the lower portion of said outer cylinder sidewall.
 33. Thesystem of claim 32 wherein said hybridization chamber device furthercomprises means for rotating said inner cylinder.
 34. The system ofclaim 33 wherein said rotating means comprises a stepper motor.
 35. Thesystem of claim 32 wherein said fluid delivery means comprises aplurality of reservoirs for storing fluids and metering pumps fordelivering fluid in selectable volumes.
 36. The system of claim 35wherein said fluid delivery means further comprises a batch heater forheating said fluids to a selected temperature before said fluids aredelivered to said chamber compartment.
 37. The system of claim 35wherein said fluid delivery means further comprises a probe concentratereservoir for storing a concentrated liquid hybridization probe and thelike, and means for mixing said concentrated hybridization probe and afluid to form a hybridization solution and delivering said hybridizationsolution to said chamber compartment in a selectable volume.
 38. Thesystem of claim 37 wherein said fluid delivery means further comprisesmeans for chilling said hybridization probe.
 39. The system of claim 32wherein said hybridization chamber device further comprises an adhesivefilm heating element for heating a fluid contained in said chambercompartment to a selected temperature, wherein said heating element iscoupled to the exterior surface of the sidewall of the outer cylinder.40. The system of claim 39 wherein said hybridization chamber devicefurther comprises a thermocouple for monitoring the temperature of fluidcontained in said chamber compartment.
 41. The system of claim 32further comprising means for fluid removal from said chamber compartmentvia valve means contained in the lower portion of said outer cylindersidewall.
 42. The system of claim 32 wherein said imaging means includesa light source for producing and delivering a beam of light of anappropriate wavelength to the membrane for exciting fluorescence of saidfluorescent product.
 43. The system of claim 42 wherein said lightsource is selected from the group consisting of a laser and atungsten-halogen lamp with a band pass filter.
 44. The system of claim43 wherein said imaging means further comprises a filter for removingundesirable wavelengths of light from said beam of light.
 45. The systemof claim 42 wherein said imaging means includes a CCD camera forrecording an image of said fluorescence emanating from said fluorescentproduct on said membrane and a filter for removing interferingwavelengths of light from said fluorescence of said fluorescent product.46. The system of claim 45 wherein said CCD camera is operable in TimeDelay and Integration mode.
 47. The system of claim 31 wherein thecontroller means is a programmable computer.
 48. A method forenzyme-linked fluorescent detection of nucleic acid comprising the stepsof: (a) attaching the nucleic acid to be detected to a membrane; (b)contacting the membrane with an oligonucleotide probe containing abinding moiety for binding an enzyme and which is able to hybridizespecifically with a complementary sequence on the nucleic acid, therebyhybridizing said probe to said nucleic acid; (c) contacting the probewith an enzyme thereby binding the enzyme to said binding moiety on theprobe; (d) contacting the enzyme with a fluorogenic substrate so thatthe enzyme converts the fluorogenic substrate into a fluorescentproduct, wherein the fluorescent product interacts with the membrane andis spatially localized thereon; and (e) illuminating said fluorescentproduct with a beam of light capable of exciting fluorescence by saidfluorescent product, wherein said fluorescence produces a pattern ofhybridization that reflects the location of said nucleic acid on saidmembrane.
 49. The method of claim 48 wherein said membrane is comprisedof nylon.
 50. The method of claim 48 wherein step (c) further comprisesattaching to said enzyme a molecule having an affinity for binding tosaid binding moiety, said molecule forming a bridge for binding of saidenzyme to said binding moiety.
 51. The method of claim 50 wherein saidbinding moiety comprises a biotin molecule covalently attached to saidprobe and said bridging molecule is selected from the group consistingof streptavidin and avidin.
 52. The method of claim 50 wherein saidbinding moiety comprises a digoxigenin molecule covalently attached tosaid probe and said bridging molecule comprises an anti-digoxigeninantibody.
 53. The method of claim 50 wherein the enzyme is alkalinephosphatase.
 54. The method of claim 53 wherein the fluorogenicsubstrate is a phosphorylated benzothiazole derivative.
 55. The methodof claim 54 wherein the fluorogenic substrate is BBTP.
 56. The method ofclaim 50 further comprising (f) imaging said pattern of hybridization byimaging means.
 57. The method of claim 56 wherein said imaging meansincludes a light source for producing and delivering a beam of light ofan appropriate wavelength to the membrane for exciting fluorescence ofsaid fluorescent product.
 58. The method of claim 57 wherein said lightsource is selected from the group consisting of a laser and atungsten-halogen lamp with a band pass filter.
 59. The method of claim57 wherein said imaging means further comprises a filter for removingundesirable wavelengths of light from said beam of light.
 60. The methodof claim 57 wherein said imaging means includes a CCD camera forrecording an image of said fluorescence emanating from said fluorescentproduct on said membrane and a filter for removing interferingwavelengths of light from said fluorescence of said fluorescent product.61. The method of claim 60 wherein said CCD camera is operable in TimeDelay and Integration mode.
 62. A hybridization chamber device forhybridizing a probe to a nucleic acid bound to a membrane comprisinggenerally horizontal, concentrically nested inner and outer cylindershaving a common axis, the inner cylinder consisting of a cylindricalsidewall having an exterior surface and sealed ends, said inner cylinderbeing rotatable about said axis, the outer cylinder consisting of astationary cylindrical sidewall having interior and exterior surfaces, atransparent window contained therein and having sealable ends, A saidinner cylinder being rotatable within said outer cylinder about saidaxis, said membrane being mounted on the exterior sidewall surface ofsaid inner cylinder; said inner and outer cylinders having diameterssuch that a there is a space between the respective sidewalls of saidinner and outer cylinders defining a chamber compartment of sufficientwidth that said inner cylinder can rotate within said outer cylinderwithout said membrane coming in contact with the interior surface ofsaid outer cylinder sidewall.
 63. The hybridization chamber device ofclaim 62 wherein said hybridization chamber device further comprisesmeans for rotating said inner cylinder.
 64. The hybridization chamberdevice of claim 63 wherein said rotating means comprises a steppermotor.
 65. The hybridization chamber device of claim 62 wherein saidhybridization chamber device further comprises a heating element forheating a fluid contained in said chamber compartment to a selectedtemperature.
 66. The hybridization chamber device of claim 69 whereinsaid hybridization chamber device further comprises a thermocouple formonitoring the temperature of a fluid contained in said chambercompartment.
 67. The hybridization chamber device of claim 65 whereinsaid heating element is a film contained on the exterior surface of thesaid wall of the outer cyliner.
 68. The hybridization chamber device ofclaim 62 further comprising means for fluid delivery, said chambercompartment being in fluid communication with said means for fluiddelivery via valve means contained in a lower portion of said outercylinder sidewall, wherein said fluid delivery means comprises aplurality of reservoirs for storing fluids and metering pumps fordelivering fluid in selectable volumes.
 69. The hybridization chamberdevice of claim 68 wherein said fluid delivery means further comprises abatch heater for heating said fluids to a selected temperature beforesaid fluids are delivered to said chamber compartment.
 70. Thehybridization chamber device of claim 68 wherein said fluid deliverymeans further comprises a probe concentrate reservoir for storing aconcentrated liquid hybridization probe and the like, and means formixing said concentrated hybridization probe and a fluid to form ahybridization solution and delivering said hybridization solution tosaid chamber compartment.
 71. The hybridization chamber device of claim70 wherein said fluid delivery means further comprises means forchilling said hybridization probe.
 72. The hybridization chamber deviceof claim 62 further comprising means for fluid removal from said chambercompartment via valve means contained in the lower portion of said outercylinder sidewall.
 73. The hybridization chamber device of claim 62wherein said hybridization chamber device further comprises imagingmeans for light delivery to said membrane and image recording offluorescence emanating from said membrane while in said hybridizationchamber device, wherein said imaging means includes a light source forproducing and delivering a beam of light of an appropriate wavelength tothe membrane for exciting fluorescence of said fluorescent product. 74.The hybridization chamber device of claim 73 wherein said light sourceis selected from the group consisting of laser and a tungsten-halogenlamp with a band pass filter.
 75. The hybridization chamber device ofclaim 74 wherein said imaging means further comprises a filter forremoving undesirable wavelengths of light from said beam of light. 76.The hybridization chamber device of claim 73 wherein said imaging meansincludes a CCD camera for recording an image of said fluorescenceemanating from said fluorescent product on said membrane and a filterfor removing interfering wavelengths of light from said fluorescence ofsaid fluorescent product.
 77. The hybridization chamber device of claim76 wherein said CCD camera is operable in Time Delay and Integrationmode.
 78. A method for detecting a macromolecule bound to a membranecomprising the steps of: (a) preparing a sample of the macromolecule andattaching the macromolecule to a membrane; (b) placing said membranecontaining said macromolecule in a chamber device of an integratedautomated imaging hybridization chamber system comprising anhybridization chamber device, means for fluid delivery to said chamberdevice, imaging means for light delivery to said membrane and imagerecording of fluorescence emanating from said membrane while in saidchamber device, and controller means for controlling operation of saidsystem; (c) introducing a probe containing a binding moiety to which anenzyme may be bound and which is able to bind specifically with saidmacromolecule into said chamber device by said fluid delivery means andbringing said probe into contact with said membrane thereby binding saidprobe to said macromolecule; (d) introducing an enzyme into said chamberdevice by said fluid delivery means and bringing said enzyme intocontact with said probe thereby binding the enzyme to said bindingmoiety on said probe; (e) introducing a fluorogenic substrate into saidchamber device by said fluid delivery means and bringing said substrateinto contact with said enzyme, wherein said enzyme converts saidfluorogenic substrate into a fluorescent product and wherein thefluorescent product interacts with the membrane and is spatiallylocalized thereon; (f) illuminating said fluorescent product in saidchamber device with a beam of light from said imaging means capable ofexciting fluorescence by said fluorescent product, wherein saidfluorescence produces a pattern of binding that reflects the location onsaid membrane of said macromolecule; and (g) imaging said pattern ofbinding by said imaging means, wherein said pattern of binding is storedas digital signals.
 79. The method of claim 78 wherein saidmacromolecule is a nucleic acid.
 80. The method of claim 79 wherein step(a) is selected from the group consisting of colony hybridization,plaque hybridization, Southern hybridization, Northern hybridization,multiplex genotyping of simple sequence repeats, sequencing byhybridization, gene mapping by hybridization, dothybridization, slothybridization, and allele-specific oligonucleotide blot hybridization.81. The method of claim 78 wherein said macromolecule is a protein. 82.The method of claim 81 wherein step (a) comprises Western blotting.